Our work will focus mainly on the synthesis of transfer RNAs including the synthesis of the modified bases in tRNAs. Dihydrouridine is the modified base present in largest amount in tRNA, and yet the mechanism of synthesis of this has not yet been worked out. We will continue our efforts to obtain a cell-free system from E. coli which can incorporate H3 either from water or from DPNH or TPNH into a modified tRNA treated with bisulfite to generate new uridines from cytidines in the dihydrouridine loop. We will also continue work on the synthesis in vitro of tRNA-containing transcripts catalyzed by the three DNA-dependent RNA polymerases of yeast separated on columns by the methods of Dr. B. Hall and his group. The hypothesis of Hall et al. that polymerase III is the enzyme specifically involved in tRNA synthesis needs to be proven experimentally. In vitro transcription experiments require DNA with a minimum of knicks and breaks, and we will continue to work on methods to obtain such DNA preparations.